Porphobilinogen (PBG) synthase catalyzes and early step in heme biosynthesis, the asymmetric addition of two molecules of amino levulinic acid to form the pyrrole porphobilinogen, and it is believed to be a primary target for lead poisoning. The primary focus of this research is on the nature of the PBG synthase metal binding site with relation to enzyme activity, Pb poisoning, and essential sulfhydryl groups. Metallo-PBG synthase containing spectroscopic probe metal ions will be prepared by a two step procedure, 1) The intrinsic ZN+2 will be removed by reversibly modifying PBG synthase with methylmethanethiosulfonate (MMTS). The resulting inactive enzyme species is mod apo-PBG synthase. 2) Probe metal ions will be used to replace Zn by reconstituting mod apo-PBG synthase in the presence of Beta mercaptoethanol, Catalytic activity can be regenerated when the probe metal ions ate Cd+2 or VO+2 making these two metal ions excellent candidates for spectroscopic study of the Zn+2 binding site by NMR (113 Cd) and EPR respectively. Spectroscopic, kinetic, and physical chemical characterization of these and other metallo PBG synthase molecules is expected to shed light on the nature of the ligands to the intrinsic metal ion and the role of that metal ion in PBG synthase catalysis. In order to probe the inhibitory PB+2 binding site, which may or may not be identical to the activating Zn+2 binding site, 207Pb PBG synthase will be prepared as described above for characterization by NMR. Kinetic and physical-chemical experiments are also proposed to study the effect of Pb+2 on PBG synthase and to determine if the Zn and Pb binding sites are identical. The fact that there are only four activating Zn+2 binding sites per octameric PBG synthase and the fact that sulfur is strongly implied as a Zn+2 ligand suggests a variety of geometric arrangements of the metal ions with regard to the quaternary structure of PBG synthase. To look at quaternary structure, an experiment is proposed to determine if the Zn+2 ions lie between subunits. The method of preparing and reconstituting mod-apo-PBG synthase (as described above) also provides fertile ground for the preparation of metallo-PBG synthase crystals for x-ray crystallographic studies. It is hoped that these studies will provide insight into the molecular mechanism of Pb poisoning and aid in the design of general therapies for heavy metal poisoning.